PROJECT SUMMARY/ABSTRACT Triple Negative Breast Cancer (TNBC) affects approximately 20% of breast cancer (BC) patients and is the most aggressive sub-type of BC, in part due to the lack of therapeutic options available for patients. Our laboratory has demonstrated that Estrogen Receptor Beta (ER?) protein is detectable at moderate to high levels in approximately 30% of TNBCs, and BC patients whose tumors retain ER? expression have enhanced therapeutic response, lower rates of breast cancer recurrence, and improved disease-free survival. In addition, we have shown that activation of ER? by estradiol (E2) decreases TNBC cell proliferation, invasion, and migration, demonstrating that ER? functions as a tumor suppressor and is a relevant and novel drug target. To determine the mechanisms by which ER? elicits its tumor suppressive effects, we have for the first time elucidated the ER? transcriptome and cistrome in TNBC cells. Through pathway analysis of our gene expression studies, we identified the NF?B pathway as one of the most suppressed pathways in response to ligand-mediated activation of ER?. Among the most down-regulated genes following E2 treatment were members of the interleukin (IL) family, which are known NF?B target genes. Chromatin Immunoprecipitation followed by sequencing (ChIPseq) for ER? in TNBC cells revealed that ER? was not only associated with EREs following E2 treatment, but was also enriched around NF?B binding sites. In fact, 12% of all ER? binding sites encode NF?B response elements. Using an NF?B reporter construct and gene expression studies, we have shown that ligand-mediated activation of ER? suppresses canonical NF?B signaling and blocks the activating functions of TNF?. We have also shown that this suppression is a result of chromatin modification. Suppression of NF?B signaling is known to inhibit proliferation and invasion of TNBC cells in vitro and decreased expression of NF?B target genes is associated with improved survival of TNBC patients, yet direct suppression of NF?B signaling has toxic side effects. However, we expect indirect or cell-specific blockade of the NF?B pathway in TNBC patients would suppress tumor progression and improve outcomes. In light of these observations and the known oncogenic properties of NF?B in TNBC, this proposal aims to address the central hypothesis that ER? elicits its tumor suppressive effects in TNBC by suppressing the NF?B signaling pathway. In Specific Aim 1, we will determine the mechanisms by which ER? suppresses NF?B signaling in TNBC using multiple cell line model systems. In Specific Aim 2, we will utilize a novel ER? transgenic mouse model to determine the impact of ER? expression and activation on TNBC progression and assess alterations in NF?B signaling during this process. Additionally, we will employ patient-derived organoids to evaluate the efficacy of ER? targeted agents and its impact on NF?B signaling. Completion of these studies will define the molecular mechanisms by which ER? suppresses NF?B signaling in TNBC and how this contributes to the anti-cancer effects of ER? and elucidate the utility of ER? targeted therapies for the treatment of TNBC.